The present invention relates to compounds having antitumor activity, in particular to new derivatives of camptothecins, processes for their preparation, their use as antitumor drugs and pharmaceutical compositions containing them as active ingredients.
Camptothecin is an alkaloid, which was isolated by Wall et al (J. Am. Chem. Soc. 83, 3888-3890 (1966)) for the first time from the tree Camptoteca acuminata, a plant originating from China, of the Nyssaceae family.
The molecule consists of a pentacyclic structure having a lactone in the ring E, which is essential for cytotoxicity.
The drug demonstrated a wide spectrum of antitumor activity, in particular against colon tumors, other solid tumors and leukemias, and the first clinical trials were performed in the early 70""s. Since Camptothecin (in the following briefly CPT) has low water solubility and in order to prepare clinical trials, the National Cancer Institute (NCI) prepared the sodium salt (NSC100880), which is water-soluble. Clinical trials in phase I and II, were not completed because of the high toxicity showed by the compound (hemorrhagic cystitis, gastrointestinal toxicity, such as nausea, vomit, diarrhoea, and myelosuppression, especially leucopenia and thrombocytopenia.
In any case, sodium salt showed a lower activity than CPT, because, at pH 7.4, the inactive form (open ring) predominates on the lactone-active one (closed ring), which predominates at pH less than 4.0.
Subsequently, many CPT analogues were synthesised in order to obtain compounds with lower toxicity and higher water solubility. Two drugs are marketed, Irinotecan (CPT-11), marketed with the Trade Mark Camptosar(copyright) by Upjohn and Topotecar(copyright), marketed with the Trade Mark Hymcamptamin(copyright) or Thycantin(copyright) , by Smith Kline and Beecham. Other derivatives are in different steps of clinical development in phase II, such as NSC-603071 (9-aminocamptothecin), 9-NC or 9-nitrocamptothecin, an oral prodrug converted in 9-aminocamptothecin, GG-211 (GI 147211), and DX-8591f, the latter being water-soluble. All the derivatives identified to-date contain the parent structure with 5 rings, essential for cytotoxicity. It was demonstrated that modifications on the first ring, such as in the case of the above-mentioned drugs increase water solubility and allow a higher tolerability of the drug.
Water-soluble Irinotecan was approved for the treatment of many solid tumors and ascites (colon-rectum, skin, stomach, breast, small and non-small cell lung, cervix and ovarian cancer and in non-Hodgkin lymphoma). Moreover, Irinotecan resulted active in solid tumors resistant to Topotecan, vincristine or melphalan and MDR-1 cells resulted marginally resistant to the drug. The active metabolite was identified as the 10-hydroxyderivatives (SN-38), produced by the action of carboxylesterases. CPT-11 showed a good activity using different administration routes, such as intraperitoneal, intravenous, oral (Costin D., Potmhexyl M. Advances in Pharmacol. 29B, 51-72 1994).
CPT-11 was administered also with cisplatin or etoposide, showing a synergistic effect, thanks to the ability to hinder DNA repair. Also in this case, however, a grade 3 and 4 leucopenia and diarrhoea arose (Sinha B. K. (1995) Topoisomerase inhibitors. Drugs 49, 11-19, 1995).
Topotecan has a significant oral bioavailability. Oral administration proved to be convenient to reach a prolonged exposition to the drug, without the use of temporary catheters being necessary (Rothenberg M. L. Annals of Oncology 8, 837-855, 1997). Also this water-soluble CPT analogue showed activity against different types of tumors, with different administration routes, intraperitoneal, intravenous, subcutaneous, oral The more promising results were obtained with Topotecan hydrochloride, intravenous infusion for 5 days, in different tumors such as small and non-small cell lung, ovarian, breast, stomach, liver, prostatae, soft tissue sarcoma, head and neck, oesophagus, resistant colon-rectum, multiform glioblastoma, chronic and acute myelocytic leukemias. However, also in this case, severe side effects occurred, such as neutropenia and thrombocytopenia, whereas gastrointestinal toxicity, such as nausea, vomit and diarrhoea were milder.
It was demonstrated that the main transformation and elimination pathways of the drug comprise lactone hydrolysis and urinary excretion: in fact, lactone form is 50% hydrolysed to open ring, 30 min after infusion. Topotecan crosses hematoencephalic barrier 10 min after infusion (30% in the cerebrospinal fluid with respect to plasma). On the contrary, camptothecin does not cross hematoencephalic barrier in significant amount, probably due to its binding with proteins.
Clinical development of 9-aminocamptothecin was hampered by its scarce water solubility. Recently, a colloidal dispersion was prepared, which made possible its entry in phase II clinical trial. Prolonged exposition (from 72 hours to 21 days) appeared to be essential to demonstrate antitumor activity, because of its short half-life (Dahut et al., 1994). Responses in patients suffering from not treated colon-rectum, and breast cancer and resistant lymphoma, were noticed. The activity demonstrated against Pgp-positive tumors suggested a lack of cross-resistance against resistant MDR-1 cells. Once again, bone marrow and gastrointestinal toxicity was observed.
Lurtotecan is the most water-soluble analogue, with an activity comparable to Topotecan in vitro. Two regimens were adopted: one 30-min infusion a day for 5 days every 3 weeks and one 72-hours infusion one time every 3 weeks. Responses in patients suffering from, neck, ovarian, breast, liver tumour were observed. Also in this case, hematic toxicity was detected. The molecule is the following: 
9-Nitrocamptothecin is an oral prodrug rapidly converted into 9-aminocamptothecin after administration. Responses were observed in patients suffering from pancreas, ovarian, and breast cancer.
Notwithstanding the major part of tumour cells is highly sensitive to topoisomerase I inhibitors, due to the high enzyme levels, some tumoral lines result to be resistant. This is due to other mechanisms, rather than the overexpression of MDR1 and MRP (multidrug resistance associated protein) genes and of their products, P (Pgp) glycoprotein and MRP protein, respectively, for which Topotecan or CPT-11 are not very good substrates, (Kawato Yet al J. Pharm. Pharmacol. 45, 444-448, (1993)).
In fact, it was observed that some resistant tumour cells contain mutant forms of topo I, accordingly the formation of the topo I-DNA complex is damaged or some cells lack in the carboxylesterase activity, necessary for converting CPT-11 in the active metabolite SN-38 and are thus resistant against this drug (Rothenberg, 1997, ibid.).
Within the drugs used in tumour therapy, the interest in inhibitors of topoisomerase I enzymes is attributed to the following considerations: a) efficacy against tumors naturally resistant to conventional drugs, topoisomerase II inhibitors included; b) the levels of the toco I enzyme remain elevated in all phases of the cycle; c) many tumors express high levels of the target enzyme; d) lack of recognition by the proteins involved in the phenomenon of multi-drug resistance (Pgp or MRP) and absence of the detoxifying enzyme-mediated metabolism, associated to the glutathione-dependent system (glutathione peroxidase and glutathione S-transferase) (Gerrits C J H., et al., Brit. J. Cancer 76, 952-962).
Once potential clinical advantages of topoisomerase I inhibitors are taken into consideration, both in terms of antitumor activity, assayed on a wide range of tumors, and the poor induction of pharmaco-resistance, the present research aims to identify topo I inhibitors with a lower toxicity with respect to the one demonstrated by the drugs on the market or in clinical phase. The factors determining the relative potency of camptothecin analogues include a) intrinsic activity of topoisomerase I inhibition; b) drug mean life; c) interaction with plasma proteins; d) the ratio between the circulating active form (lactone) and the non active one (carboxylate); e) drug sensitivity relative to cell outflow mediated by glycoprotein P or MRP; f) bond stability with topoisomerase I (Rothenberg, 1997, ibid.).
Among the main adverse effects of Irinotecan and other camptothecins derivatives, myelosuppression and gastrointestinal toxicity, such as diarrhoea and vomit, have been observed. Diarrhoea can have an early or late onset and can be a dose-limiting factor. Vomit and late diarrhoea are induced by many antitumor drugs, while early diarrhoea occurring during or immediately after infusion is almost specific for Irinotecan and some camptothecin derivatives.
Toxic effects occur mainly in the intestinal tract.
In order to reduce diarrhoea, CPT-11 was administered in some clinical trials, in combination with loperamide, a synthetic oppioid, agonist of the mu-oppioid enteric receptors (Abigerges, 1994; Abigerges, 1995), as well as with an inhibitor of the enkephalinases (acetorfan) or with ondansetron, an antagonist of the 5-HT3 receptors, or with diphenidramine, an antagonist of H1 receptors.
To date, the problems connected with the use of camptothecin derivatives as antitumor drugs can be summarised in the following items:
camptothecin (CPT), and many of its active derivatives have low water solubility;
the subsequent derivatives are endowed with severe side effects at gastrointestinal and bone marrow level;
some tumour lines developed resistance against topoisomerase I inhibitors;
there is the constant search for a better therapeutic index.
Patent application WO97/31003, herein incorporated for reference, discloses derivatives of camptothecins substituted at positions 7, 9 and 10. Position 7 provides the following substitutions: xe2x80x94CN, xe2x80x94CH(CN)xe2x80x94R4, xe2x80x94CHxe2x95x90C(CN)xe2x80x94R4, xe2x80x94CH2xe2x80x94CHxe2x95x90C(CN)xe2x80x94R4, xe2x80x94C(xe2x95x90NOH)xe2x80x94NH2, xe2x80x94CHxe2x95x90C(NO2)xe2x80x94R4, xe2x80x94CH(CN)xe2x80x94R5, xe2x80x94CH(CH2NO2)xe2x80x94R5, 5-tetrazolyl, 2 -(4,5-dihydroxazolyl), 1,2,4-oxadiazolidin-3-yl-5-one, wherein R4 is hydrogen, linear or branched alkyl from 1 to 6 carbon atoms, nitrile, carboxyalkoxy. Of these possible compounds, WO97/31003 enables the disclosure only of camptothecin derivatives bearing at position 7 the group xe2x80x94CN and xe2x80x94CHxe2x95x90C(CN)2, with unsubstituted positions 9 and 10.
Of these compounds, the best one proved to be the 7-nitrile (R4=xe2x80x94CN), hereinafter named CPT 83, with cytotoxic activity on non-small cells lung carcinoma (non-SCLC, H-460). This tumour line is intrinsically resistant to cytotoxic therapy and is only moderately responsive to topoisomerase I inhibitors, notwithstanding the overexpression of the target enzyme. CPT 83 is more active than Topotecan, taken as reference compound and on the whole it offers a better pharmacological profile, even in terms of tolerability, then a better therapeutic index.
CPT 83 is prepared through a synthesis route comprising the oxidation of 7-hydroxymethylcamptothecin to camptothecin 7-aldehyde, the transformation of the latter into oxime and final conversion into nitrile.
The starting compound and the intermediates are disclosed in Sawada et al Chem. Pharm. Bull. 39, (10) 2574 (1991). This paper makes reference to a patent family with priority of 1981, for example European patent application EP 0056692, published in 1982, incorporated herein for reference. In these publications there are disclosed, among others, the compounds camptothecin 7-aldehyde and its oxime. The usefulness of these derivatives is to provide compounds with antitumor activity having low toxicity starting from 7-hydroxymethylcamptothecin. In the paper published on Chem. Pharm. Bull. 39, (10) 2574 (1991), the authors demonstrate that, with respect to camptothecin, the 7-alkyl and 7-acyloxymethyl derivatives, which were not foreseen in the above mentioned patent application, are the more active compounds on lines of murine leukemia L12 10, while lower activity, always with respect to camptothecin, was observed in compounds bearing 7-substitutions with high polar character, such as hydrazones and the oxime xe2x80x94CH(xe2x95x90NOH).
It has now surprisingly been found that camptothecins bearing an alkyloxime O-substituted at position 7 are endowed with antitumor activity higher than the compound of reference Topotecan. More surprisingly, it has been found that camptothecins bearing an enamino group on position 7, are also endowed with antitumor activity. Said compounds have better therapeutic index.
Accordingly, it is an object of the present invention compounds of general formula (I): 
wherein: R1 is a xe2x80x94C(R5)xe2x95x90Nxe2x80x94O(n)R4 group, wherein R4 is hydrogen or a C1-C8 linear or branched alkyl or C1-C8 linear or branched alkenyl group or C3-C10 cycloalkyl, or (C3-C10) cycloalkyl xe2x80x94(C1-C8) linear or branched alkyl group, or C6-C14 aryl, or (C6-C14) aryl xe2x80x94(C1-C8) linear or branched alkyl group, or a heterocyclic or heterocyclo xe2x80x94(C1-C8) linear or branched alkyl group, said heterocyclic group containing at least one heteroatom selected from the group consisting of nitrogen atom, optionally substituted with a (C1-C8) alkyl group, and/or oxygen and/or sulfur; said alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aryl-alkyl, heterocyclic or heterocyclo-alkyl groups, being optionally substituted with one or more groups selected from the group consisting of: halogen, hydroxy, keto, C1-C8 alkyl, C1-C8 alkoxy, phenyl, cyano, nitro, xe2x80x94NR6R7, wherein R6 and R7, the same or different between them, are hydrogen, (C1-C8) linear or branched alkyl; the xe2x80x94COOH group or a pharmaceutically acceptable ester thereof; or the -CONR8R9 group, wherein R8 and R9, the same or different between them, are hydrogen, (C1-C8) linear or branched alky, phenyl; or
R4 is a (C6-C10) aroyl or (C6-C10) arylsulfonyl group, optionally substituted with one or more groups selected from the group consisting of: halogen, hydroxy, C1-C8 linear or branched alky, C1-C8 linear or branched alkoxy, phenyl, cyano, nitro, xe2x80x94NR10R11, wherein R10 and R11, the same or different between them are hydrogen, C1-C8 linear or branched alkyl;
R4 is a polyaminoalkyl group; or
R4 is a glycosyl group;
n is the number 0 or 1;
R5 is hydrogen, C1-C8 linear or branched alkyl, C1-C8 linear or branched alkenyl, C3-C10 cycloalkyl, (C3-C10) cycloalkyl xe2x80x94(C1-C8) linear or branched alkyl, C6-Cl14 aryl, (C6-C14) aryl xe2x80x94(C1-C8) linear or branched alkyl;
R2 and R3, the same or different between them are hydrogen, hydroxy, C1-C8 linear or branched alkoxy;
their N1-oxides, their single isomers, in particular the syn and anti isomers of the xe2x80x94C(R5)xe2x95x90Nxe2x80x94O(n)R4 group, their possible enantiomers, diastereoisomers and relative mixtures, the pharmaceutically acceptable salts thereof and their active metabolites;
with the proviso that when R5, R2 and R3 are hydrogen and n is 1, then R4 is different from hydrogen.
The present invention comprises the use of the compounds of the above-mentioned formula (I) as active ingredients for medicaments, in particular for medicaments useful for the treatment of tumors. A further object of the present invention is also the use of the compounds of formula (I) as active ingredients for medicaments useful for treating viral infections. Another object of the present invention is also the use of the compounds of formula (I) as active ingredients for medicaments having antiplasmodium falciparum activity.
The present invention comprises pharmaceutical compositions containing compounds of formula (I) as active ingredients, in admixture with pharmaceutically acceptable vehicles and excipients.
The present invention comprises also processes for the preparation of compounds of formula (I), and the relative key intermediates.